PROJECT SUMMARY/ABSTRACT Prostate-specific membrane antigen (PSMA) is the preeminent enzyme-biomarker for prostate cancer because it is expressed on nearly all prostate cancers and increased expression correlates with progression to castration resistance and metastatic disease. In addition, the endothelial expression of PSMA on the neovasculature of non-prostatic tumors offers a broader opportunity for the delivery of chemotherapy and other agents to a variety of other vascularized solid tumors. We recently demonstrated that the addition of an albumin-binding motif to PSMA-targeted agents dramatically slows clearance and allows for tumor uptake of nearly 50% of an injected dose. Our irreversible- binding phosphoramidate targeting molecules selectively target and rapidly penetrate prostate tumor cells via internalization of the PSMA enzyme-inhibitor complex into endosomes/lysosomes. In addition, our novel Phos- Am linker system is stable at physiologically pH, but can be tuned to release amine-containing drugs at endosomal pH. Based on these 3 key achievements, we are now positioned to develop the first effective PSMA-targeted SMDC, which is the objective of this application. Our central hypothesis is that PSMA-targeted SMDCs that harness the affinity and cell-penetrating properties of our irreversible PSMA inhibitors in consort with our tunable pH-sensitive cleavable Phos-Am linker scaffold will be effective against PSMA+ tumors. We will test our central hypothesis and accomplish the objective of this application by pursuing the following specific aims. Aim #1: Compare the stability and in vitro performance of a Phos-Am and Val-Cit linked PSMA- targeted SMDC. We will examine the in vivo cytotoxicity of our Phos-Am(HoSer) and the Val-Cit linked PSMA- targeted SMDC. The stability of these conjugates will be assessed in buffer (pH = 4.5-7.4), in the presence of cathepsin B, as well as mouse and human plasma. Concentration-dependent in vitro cytotoxicity will be assessed with PSMA(+) and PSMA(-) cells lines. Aim #2: Compare the clearance kinetics and in vivo performance of three known albumin binding motifs. We will compare the clearance and in vivo performance of the PSMA-targeted SMDCs with and without albumin-binding motifs (L-AlbuTag and Evans Blue Dye). The top candidate from Aim #1 will be advanced to Aim #2 to include an albumin moiety and the SMDC which demonstrates the highest tumor uptake will be advanced to in vivo efficacy using the well-established PC3-PSMA tumor xenografts in a mouse model. The expected outcome of these studies is a PSMA-targeted SMDC positioned for pre-clinical studies in a subsequent proposal. Employing an irreversible-binding small-molecule ligand to PSMA combined with a novel pH-triggered linker, along with optimal clearance kinetics provided by an albumin-binding motif is highly novel.